The present invention relates to a method for the preparation of alpha(.alpha.)-L-aspartyl-L-phenylalanine methyl ester hydrochloride (.alpha.-APM(HCl)) which is used to prepare .alpha.-L-aspartyl-L-phenylalanine methyl ester (.alpha.-APM), a sweetening agent which is approximately 200 times sweeter than sucrose. The potency of this compound, a dipeptide, enables one to sweeten foods and beverages using a far lesser amount than one could with sugar. Consequently, it has enabled millions of consumers to reduce their caloric intake while not having to give up the sweet things in life. It also lacks the unpleasant aftertaste associated with other sweeteners such as saccharin and cyclamate. Additionally, the present invention relates to a method of increasing the .alpha./B ratio of APM (HCl) and methods of producing a reaction mixture of N-formyl-.alpha.-L-aspartyl-L-phenylalanine (FAP) having a stirrable viscosity.
.alpha.-APM is not new and was described in U.S. Pat. 3,492,131 to Schlatter in 1970. Numerous other patents involving different methods of manufacture and related compounds have since issued and much literature has been written heralding the effect the dipeptide has had on the low calorie sweetener industry. Up until the present time, however, the methods of preparation have involved costly isolation and recovery processes which consequently must be shouldered by the consumer. The present invention is a method of preparation having the operational simplicity of a one-pot process as well as improved yields of the desired end product.
.alpha.-L-aspartyl-L-phenylalanine methyl ester is a dipeptide composed essentially of two amino acids, L-aspartic acid and L-phenylalanine. It has been known for some time that the sweetening property of the dipeptide is dependent upon the stereochemistry of these individual amino acids. Each of these amino acids can exist in either the D or L form, and it has been determined that the L-aspartyl-L-phenylalanine esters are sweet while the corresponding D-D, D-L and L-D isomers are not. Combinations of the isomers which contain the L--L dipeptide, DL-aspartyl-L phenylalanine, L-aspartyl DL-phenylalanine and DL-aspartyl-DL phenylalanine are sweet, but only half as sweet since the racemate contains 1/2 of the L--L moiety.
The dipeptide is produced through a coupling reaction in which L-aspartic acid is joined with L-phenylalanine or its methyl ester. This coupling reaction requires an amino protecting group attached to the aspartic acid moiety such as formyl, acetyl, acetoacetyl, benzyl, substituted and unsubstituted carbobenzoxy, t-butoxy carbonyl and the hydrohalide salt. The amino protecting group, often referred to in the art as the N-protecting group, for purposes of this disclosure shall be referred to as N-formyl since the formyl moiety is the blocking agent of the present invention. N-formyl aspartic anhydride is a widely used starting material and its process has been described extensively. See U.S. Pat. No. 4,173,562.
However, obtaining isolated N-formyl aspartic anhydride has resulted in relatively low yields when a large excess of formic acid is used as solvent. A twofold excess of formic acid, for example, creates the need for addition of toluene or other solvents to reduce anhydride solubility, thereby requiring extensive solvent recovery steps.
The coupling reaction is carried out in a solvent and is a common step in several patented processes for the production of .alpha.-L-aspartyl-L-phenylalanine methyl ester (.alpha.-APM); see U.S. Pat. No. 3,962,207 to Uchiyama, U.S. Pat. No. 4,173,562 to Bachman and EPO Patent 127,411 to Yaichi et al., all of which are incorporated herein by reference. During the coupling reaction of the two amino acids, two isomers are produced as intermediates and their stereochemistry ultimately determines the sweetness of the particular molecule. The alpha (.alpha.) isomer is the desired product in that isolated fractions of pure .alpha.-APM possess a sweetness about 130-200 times that of sugar. The beta (.beta.) isomer fraction, however, has no such sweetness.
This invention is directed to improvements in the preparation of .alpha.-APM (HCl) which result in lower costs of production and increased yields of the .beta. isomer which is the desired end product.
The .alpha. and .beta. isomers of APM are given below: ##STR1##
It has been determined that formation of the .alpha. and .beta. isomers and their respective ratios from the coupling reaction depends upon what kind of solvent is used to carry out the reaction, the temperature at which the reaction occurs and the quantity of the solvents used. According to U.S. Pat. No. 4,173,562 to Bachman, an .alpha./.beta. isomer ratio of 75:25 is achievable when acetic acid is used as a solvent in the coupling reaction at 50.degree. C. The molar ratio of acetic acid to phenylalanine must be at least 10:1. The .alpha./.beta. isomer ratio drops considerably to 69/31 when the acetic acid to L-phenylalanine molar ratio is reduced to 6:1. The present invention shows that the .alpha./.beta. ratio can be increased to about 80/20 if the acetic acid, used as a solvent in the coupling reaction is partially replaced with an alkyl ester, hindered alcohol or mixture thereof. For purposes of this disclosure, hindered alcohol as it is used herein shall mean a secondary or tertiary alcohol.
A problem that resides with the use of these solvents in this process is that after 0.5-3 hours of reaction time, the reaction mixture solidifies and becomes substantially difficult to agitate, concentrate, deformylate, or remove from a reactor. A stirrable system is necessary for at least two reasons. First, stirring ensures a mixing of the reactants to achieve a complete reaction. Secondly, solvent must later be removed by distillation.
Another problem that exists in the prior art is that under some techniques, 25% or more of the .alpha.-APM is lost because it remains in the original reaction solution. See U.S. Pat. No. 4,173,562. A further problem is that in the '562 patent, N-formyl-L-aspartic anhydride is produced from a reaction mixture of aspartic acid, a large excess of formic acid and acetic anhydride. The excess amount of formic acid must at some point be removed by distillation and separated from acetic acid which adds to the cost of the final product.
U.S. Pat. No. 3,962,207 describes a similar process in which L-aspartic anhydride hydrochloride is coupled with L-phenylalanine methyl ester. A problem that arises in the '207 process is that a large amount of L-phenylalanine methyl ester is required which adds to the cost of the process. Secondly, that results in the formation of significant amounts of tri-peptides which must be removed and thereby necessitate expensive and elaborate separation techniques. This is not required in the present invention.
U.S. Ser. No. 156,268 describes a one-pot process for preparation of .alpha.-APM in which reaction by-products which result from the formation of formylated L-aspartic anhydride also serve as the solvent for the coupling reaction with L-phenylalanine.